Protocol 31645: Probe Assay - Kcnk16<em1(IMPC)J>
Version 1.0

Notes

Taqman qPCR protocols are run on a real time PCR instrument. Use an appropriate instrument specific Fluorophore/Quencher combination.

 

The genotyping protocol(s) presented here have been optimized for reagents and conditions used by The Jackson Laboratory (JAX). To genotype animals, JAX recommends researchers validate the assay independently upon receipt of animals into their facility. Reaction cycling temperature and times may require additional optimization based on the specific genotyping reagents used.

Expected Results

>chr14:20265407-20265506 100bp TCTGCATACTACCTCCTTAGGC TCCGAGGTTCTGGCTAAGAC

Mutant=  101 bp

Wild Type = 100 bp

Sequence

WT Region:  CTCTCTCTCTCTCTCTCTCTCTCTCTCTCTCTCTCTGTGTGTGTGTGTGTGTGCGTGATATTTTCCTAGCTATCAAGAGCATCTGCATACTACCTCCTTAGGCCCTGGCTTCCcactggctcattagagcacacacttcagtcaaccgctggccacttagtgtcttagccagaacctcggatccttcatgtccatttctgtgaacaggaatgatgctatctaagagacaggaaagcatcctcctgagagcttggtgacaggctgcactcagtaactgctgagcctgctgtccgtgcttcttccacaggtcatcctggaagcctgggtgaagggtgtgaaccccaaaggcaactccaccaatcccagcaactgggacttcgggagcagcttcttctttgcaggcacagtggtcaccaccataggtaagtgggagaggagtgaggcctcccttggccctgctgagcttggggtccattctaaatgccactgctggtctgggaatatgctttatccCGGAGGTGACAGACAGGGCACAGGTTGGGAAGTGGGTAGGTTGGCTGCAGCTGTAGTTGCCTGGACTCACTTCTCTGGGTCACACTGAAGGGTTAGAAAGAGCTATTCCTCAGTACA

 

Deleted Sequence cactggctcattagagcacacacttcagtcaaccgctggccacttagtgtcttagccagaacctcggatccttcatgtccatttctgtgaacaggaatgatgctatctaagagacaggaaagcatcctcctgagagcttggtgacaggctgcactcagtaactgctgagcctgctgtccgtgcttcttccacaggtcatcctggaagcctgggtgaagggtgtgaaccccaaaggcaactccaccaatcccagcaactgggacttcgggagcagcttcttctttgcaggcacagtggtcaccaccataggtaagtgggagaggagtgaggcctcccttggccctgctgagcttggggtccattctaaatgccactgctggtctgggaatatgctttatcc

400 bp deletion beginning at Chromosome 14 positive strand position 20,265,075 bp and ending after 20,265,474 bp (GRCm38/mm10).

JAX Protocol

Protocol Primers

Primer 5' Label Sequence 5' → 3' 3' Label Primer Type Reaction Note
37333 TCT GCA TAC TAC CTC CTT AGG C Common A
37334 TCC GAG GTT CTG GCT AAG AC Wild type Reverse A
37335 TGA GTC CAG GCA ACT ACA GC Mutant Reverse A
37336 Fluorophore-1 CAG TCA ACC GCT GGC C Quencher-1 WT Probe
37337 Fluorophore-2 AGA CAG GGC ACA GGT TGG Quencher-2 MUT Probe

Reaction A

Component Final Concentration
Kapa Probe Fast QPCR 1.00 X
ddH2O
37333 0.40 uM
37334 0.40 uM
37335 0.40 uM
Wt Probe 0.15 uM
Mutant Probe 0.15 uM
DNA

Cycling

Step Temp °C Time Note
1 95.0 --
2 95.0 --
3 60.0 --
4 -- repeat steps 2-3 for 40 cycles
5 40.0 -- Forever
JAX uses a very high speed Taq (~1000 bp/sec), use cycling times recommended for your reagents.

Strains Using This Protocol

This is the only strain that uses this protocol.